Modulation of diacylglycerol acyltransferase 1 expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of diacylglycerol acyltransferase 1. The compositions comprise oligonucleotides, targeted to nucleic acid encoding diacylglycerol acyltransferase 1. Methods of using these compounds for modulation of diacylglycerol acyltransferase 1 expression and for diagnosis and treatment of disease associated with expression of diacylglycerol acyltransferase 1, such as obesity and obesity-related conditions, are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.12/173,744, filed Jul. 15, 2008, which is a continuation of U.S.application Ser. No. 10/803,482, filed Mar. 18, 2004, which is acontinuation-in-part application of U.S. application Ser. No. 10/394,808filed Mar. 21, 2003, each of which is incorporated herein by referencein its entirety.

INCORPORATION OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0003USC2SEQ.txt, created on Mar. 7, 2012 which is 60 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Triglycerides are one of the major energy storage molecules ineukaryotes. The absorption of triglycerides (also calledtriacylglycerols) from food is a very efficient process that occurs by aseries of steps wherein the dietary triacylglycerols are hydrolyzed inthe intestinal lumen and then resynthesized within enterocytes. Theresynthesis of triacylglycerols can occur via the monoacylglycerolpathway, which commences with monoacylglycerol acyltransferase (MGAT)catalyzing the synthesis of diacylglycerol from monoacylglycerol andfatty acyl-CoA. An alternative synthesis of diacylglycerols is providedby the glycerol-phosphate pathway, which describes the coupling of twomolecules of fatty acyl-CoA to glycerol-3-phosphate. In either case,diacylglycerol is then acylated with another molecule of fatty acyl-CoAin a reaction catalyzed by one of two diacylglycerol acyltransferaseenzymes to form the triglyceride.

The reaction catalyzed by diacylglycerol acyltransferase 1 is the finaland only committed step in triglyceride synthesis. As such,diacylglycerol acyltransferase 1 is involved in intestinal fatabsorption, lipoprotein assembly, regulating plasma triglycerideconcentrations, and fat storage in adipocytes. Although identified in1960, the genes encoding human and mouse diacylglycerol acyltransferase1 (also called DGAT1, acyl CoA:diacylglycerol acyltransferase, acylCoA:cholesterol acyltransferase-related enzyme, ACAT related geneproduct, and ARGP1) were not cloned until 1998. U.S. Pat. No. 6,100,077refers to an isolated nucleic acid encoding a human diacylglycerolacyltransferase 1. Diacylglycerol acyltransferase 1 is a microsomalmembrane bound enzyme and has 39% nucleotide identity to the relatedacyl CoA:cholesterol acyltransferase. A splice variant of diacylglycerolacyltransferase 1 has also been cloned that contains a 77 nucleotideinsert of unspliced intron with an in-frame stop codon, resulting inatruncated form of diacylglycerol acyltransferase 1 that terminates atArg-387 deleting 101 residues from the C-terminus containing theputative active site.

Dysregulation of diacylglycerol acyltransferase 1 may play a role in thedevelopment of obesity. Upon differentiation of mouse 3T3-L1 cells intomature adipocytes, a 90 fold increase in diacylglycerol acyltransferase1 levels is observed. However, forced overexpression of diacylglycerolacyltransferase 1 in mature adipocytes results in only a 2 fold increasein diacylglycerol acyltransferase 1 levels. This leads to an increase incellular triglyceride synthesis without a concomitant increase intriglyceride lipolysis, leading to the suggestion that manipulation ofthe steady state level of diacylglycerol acyltransferase 1 may offer apotential means to treat obesity.

Alterations in diacylglycerol acyltransferase 1 expression may affecthuman body weight. In a random Turkish population, five polymorphisms inthe human diacylglycerol acyltransferase 1 promoter and 5′ non-codingsequence have been identified. One common variant, C79T, revealedreduced promoter activity for the 79T allele and is associated with alower body mass index, higher plasma cholesterol HDL levels, and lowerdiastolic blood pressure in Turkish women.

Diacylglycerol acyltransferase 1 knockout mice exhibit interestingphenotypes which indicate that inhibition of diacylglycerolacyltransferase 1 may offer a strategy for treating obesity andobesity-associated insulin resistance. Mice lacking diacylglycerolacyltransferase 1 are viable and can still synthesize triglyceridesthrough other biological routes. However the mice are lean and resistantto diet-induce obesity, have decreased levels of tissue triglycerides,and increased sensitivity to insulin and leptin.

Currently, there are no known therapeutic agents which effectivelyinhibit the synthesis of diacylglycerol acyltransferase 1 and to date,investigative strategies aimed at modulating diacylglycerolacyltransferase 1 function have involved naturally-occurring smallmolecule derivatives of roselipins and xanthohumols isolated fromGliocladium roseum and Humulus lupulus, respectively.

Consequently, there remains a long felt need for additional agentscapable of effectively inhibiting diacylglycerol acyltransferase 1function.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of diacylglycerol acyltransferase 1. In particular, thisinvention relates to compounds, particularly oligonucleotide compounds,which, in preferred embodiments, hybridize with nucleic acid moleculesencoding diacylglycerol acyltransferase 1. Such compounds are shownherein to modulate the expression of diacylglycerol acyltransferase 1.

The present invention is directed to compounds, especially nucleic acidand nucleic acid-like oligomers, which are targeted to a nucleic acidencoding diacylglycerol acyltransferase 1, and which modulate theexpression of diacylglycerol acyltransferase 1. Pharmaceutical and othercompositions comprising the compounds of the invention are alsoprovided.

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products and is uniquely useful in a numberof therapeutic, diagnostic, and research applications for the modulationof diacylglycerol acyltransferase 1 expression.

Further provided are methods of screening for modulators ofdiacylglycerol acyltransferase 1 and methods of modulating theexpression of diacylglycerol acyltransferase 1 in cells, tissues oranimals comprising contacting said cells, tissues or animals with one ormore of the compounds or compositions of the invention. In thesemethods, the cells or tissues may be contacted in vivo. Alternatively,the cells or tissues may be contacted ex vivo.

Methods of treating an animal, particularly a human, suspected of havingor being prone to a disease or condition associated with expression ofdiacylglycerol acyltransferase 1 are also set forth herein. Such methodscomprise administering a therapeutically or prophylactically effectiveamount of one or more of the compounds or compositions of the inventionto the person in need of treatment.

Also provided is a method of making a compound of the inventioncomprising specifically hybridizing in vitro a first nucleobase strandcomprising a sequence of at least 8 contiguous nucleobases of thesequence set forth in SEQ ID NO: 4 to a second nucleobase strandcomprising a sequence sufficiently complementary to said first strand soas to permit stable hybridization.

The invention further provides a compound of the invention for use intherapy.

The invention further provides use of a compound or composition of theinvention in the manufacture of a medicament for the treatment of anyand all conditions disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION A. Overview of the Invention

The present invention employs compounds, preferably oligonucleotides andsimilar species for use in modulating the function or effect of nucleicacid molecules encoding diacylglycerol acyltransferase 1. This isaccomplished by providing oligonucleotides that specifically hybridizewith one or more nucleic acid molecules encoding diacylglycerolacyltransferase 1. As used herein, the terms “target nucleic acid” and“nucleic acid molecule encoding diacylglycerol acyltransferase 1” havebeen used for convenience to encompass DNA encoding diacylglycerolacyltransferase 1, RNA (including pre-mRNA and mRNA or portions thereof)transcribed from such DNA, and also cDNA derived from such RNA. Thehybridization of a compound of this invention with its target nucleicacid is generally referred to as “antisense”. Consequently, thepreferred mechanism believed to be included in the practice of somepreferred embodiments of the invention is referred to herein as“antisense inhibition.” Such antisense inhibition is typically basedupon hydrogen bonding-based hybridization of oligonucleotide strands orsegments such that at least one strand or segment is cleaved, degraded,or otherwise rendered inoperable. In this regard, it is presentlypreferred to target specific nucleic acid molecules and their functionsfor such antisense inhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA. One preferred result of such interferencewith target nucleic acid function is modulation of the expression ofdiacylglycerol acyltransferase 1. In the context of the presentinvention, “modulation” and “modulation of expression” mean either anincrease (stimulation) or a decrease (inhibition) in the amount orlevels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA.Inhibition is often the preferred form of modulation of expression andmRNA is often a preferred target nucleic acid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases that pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases that can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termsthat are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of an antisense compoundcan be, but need not be, 100% complementary to that of its targetnucleic acid to be specifically hybridizable. Moreover, anoligonucleotide may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridizationevent (e.g., a loop structure or hairpin structure). Preferably, theantisense compounds comprise at least 8 contiguous nucleobases of anantisense sequence disclosed herein. It is preferred that the antisensecompounds of the present invention comprise at least 70%, or at least75%, or at least 80%, or at least 85% sequence complementarity to atarget region within the target nucleic acid. In another embodiment, theantisense compounds of the present invention comprise 90% sequencecomplementarity and even more preferably comprise 95% or at least 99%sequence complementarity to the target region within the target nucleicacid sequence to which they are targeted. For example, an antisensecompound in which 18 of 20 nucleobases of the antisense compound arecomplementary to a target region, and would therefore specificallyhybridize, would represent 90 percent complementarity. In this example,the remaining noncomplementary nucleobases may be clustered orinterspersed with complementary nucleobases and need not be contiguousto each other or to complementary nucleobases. As such, an antisensecompound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the present invention. Percent complementarity of anantisense compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656).

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome preferred embodiments, homology, sequence identity orcomplementarity, between the oligomeric and target is between about 50%to about 60%. In some embodiments, homology, sequence identity orcomplementarity, is between about 60% to about 70%. In preferredembodiments, homology, sequence identity or complementarity, is betweenabout 70% and about 80%. In more preferred embodiments, homology,sequence identity or complementarity, is between about 80% and about90%. In some preferred embodiments, homology, sequence identity orcomplementarity, is about 90%, about 92%, about 94%, about 95%, about96%, about 97%, about 98%, or about 99%.

B. Compounds of the Invention

According to the present invention, compounds include antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds that hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, circular orhairpin oligomeric compounds and may contain structural elements such asinternal or terminal bulges or loops. Once introduced to a system, thecompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While the preferred form of antisense compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, induces potent and specific antisense-mediated reduction ofthe function of a gene or its associated gene products. This phenomenonoccurs in both plants and animals and is believed to have anevolutionary connection to viral defense and transposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611-620).

The primary interference effects of dsRNA are posttranscriptional(Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507).The posttranscriptional antisense mechanism defined in Caenorhabditiselegans resulting from exposure to double-stranded RNA (dsRNA) has sincebeen designated RNA interference (RNAi). This term has been generalizedto mean antisense-mediated gene silencing involving the introduction ofdsRNA leading to the sequence-specific reduction of endogenous targetedmRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, thesingle-stranded RNA oligomers of antisense polarity of the dsRNAs havebeen reported to be the potent inducers of RNAi (Tijsterman et al.,Science, 2002, 295, 694-697).

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

The oligonucleotides of the present invention also include modifiedoligonucleotides in which a different base is present at one or more ofthe nucleotide positions in the oligonucleotide. For example, if thefirst nucleotide is an adenosine, modified oligonucleotides may beproduced which contain thymidine, guanosine or cytidine at thisposition. This may be done at any of the positions of theoligonucleotide. These oligonucleotides are then tested using themethods described herein to determine their ability to inhibitexpression of diacylglycerol acyltransferase 1 mRNA.

While oligonucleotides are a preferred form of the compounds of thisinvention, the present invention comprehends other families of compoundsas well, including but not limited to oligonucleotide analogs andmimetics such as those described herein.

The compounds in accordance with this invention preferably comprise fromabout 8 to about 80 nucleobases (i.e. from about 8 to about 80 linkednucleosides). One of ordinary skill in the art will appreciate that theinvention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

In one preferred embodiment, the compounds of the invention are 12 to 50nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases inlength.

In another preferred embodiment, the compounds of the invention are 15to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

Particularly preferred compounds are oligonucleotides from about 12 toabout 50 nucleobases, even more preferably those comprising from about15 to about 30 nucleobases.

Antisense compounds 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases).Exemplary compounds of this invention may be found identified in theExamples and listed in Tables 1, 2, 4, 10, and 11. One having skill inthe art armed with the preferred antisense compounds illustrated hereinwill be able, without undue experimentation, to identify furtherpreferred antisense compounds.

C. Targets of the Invention

“Targeting” an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes diacylglycerolacyltransferase 1.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes, having translation initiation codons withthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and5′-CUG, have been shown to function in vivo. Thus, the terms“translation initiation codon” and “start codon” can encompass manycodon sequences, even though the initiator amino acid in each instanceis typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA transcribed from a gene encodingdiacylglycerol acyltransferase 1, regardless of the sequence(s) of suchcodons. It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions that may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsopreferred to target the 5′ cap region.

Accordingly, the present invention provides antisense compounds thattarget a portion of nucleotides 1-1976 as set forth in SEQ ID NO: 4. Inone embodiment, the antisense compounds target at least an 8 nucleobaseportion of nucleotides 1-1976 as set forth in SEQ ID NO: 4. In anotherembodiment, the antisense compounds target at least an 8 nucleobaseportion of nucleotides 1-244 comprising the 5′UTR as set forth in SEQ IDNO: 4. In another embodiment, the antisense compounds target at least an8 nucleobase portion of nucleotides 1712-1976 comprising the 3′UTR asset forth in SEQ ID NO: 4. In another embodiment, the antisensecompounds target at least an 8 nucleobase portion of nucleotides245-1711 comprising the coding region as set forth in SEQ ID NO: 4. Instill other embodiments, the antisense compounds target at least an 8nucleobase portion of a “preferred target segment” (as defined herein)as set forth in Table 3.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

Alternative RNA transcripts can be produced from the same genomic regionof DNA. These alternative transcripts are generally known as “variants”.More specifically, “pre-mRNA variants” are transcripts produced from thesame genomic DNA that differ from other transcripts produced from thesame genomic DNA in either their start or stop position and contain bothintronic and exonic sequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

Variants can be produced through the use of alternative signals to startor stop transcription. Pre-mRNAs and mRNAs can possess more than onestart codon or stop codon. Variants that originate from a pre-mRNA ormRNA that use alternative start codons are known as “alternative startvariants” of that pre-mRNA or mRNA. Those transcripts that use analternative stop codon are known as “alternative stop variants” of thatpre-mRNA or mRNA. One specific type of alternative stop variant is the“polyA variant” in which the multiple transcripts produced result fromthe alternative selection of one of the “polyA stop signals” by thetranscription machinery, thereby producing transcripts that terminate atunique polyA sites. Within the context of the invention, the types ofvariants described herein are also preferred target nucleic acids.

The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid that are accessible forhybridization.

While the specific sequences of certain preferred target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional preferred target segments may beidentified by one having ordinary skill.

Target segments 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). One having skill in the art armed with the preferredtarget segments illustrated herein will be able, without undueexperimentation, to identify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

The oligomeric compounds are also targeted to or not targeted to regionsof the target diacylglycerol acyltransferase 1 nucleobase sequence(e.g., such as those disclosed in Example 13) comprising nucleobases1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800,801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150,1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450,1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750,1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-1976 of thediacylglycerol acyltransferase 1 sequence, or any combination thereof.

In one embodiment, the oligonucleotide compounds of this invention are100% complementary to these sequences or to small sequences found withineach of the above listed sequences. Preferably, the antisense compoundscomprise at least 8 contiguous nucleobases of an antisense compounddisclosed herein. In another embodiment the oligonucleotide compoundshave from at least 3 or 5 mismatches per 20 consecutive nucleobases inindividual nucleobase positions to these target regions. Still othercompounds of the invention are targeted to overlapping regions of theabove-identified portions of the diacylglycerol acyltransferase 1sequence.

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of diacylglycerol acyltransferase 1.“Modulators” are those compounds that decrease or increase theexpression of a nucleic acid molecule encoding diacylglycerolacyltransferase 1 and which comprise at least an 8-nucleobase portionthat is complementary to a preferred target segment. The screeningmethod comprises the steps of contacting a preferred target segment of anucleic acid molecule encoding diacylglycerol acyltransferase 1 with oneor more candidate modulators, and selecting for one or more candidatemodulators which decrease or increase the expression of a nucleic acidmolecule encoding diacylglycerol acyltransferase 1. Once it is shownthat the candidate modulator or modulators are capable of modulating(e.g. either decreasing or increasing) the expression of a nucleic acidmolecule encoding diacylglycerol acyltransferase 1, the modulator maythen be employed in further investigative studies of the function ofdiacylglycerol acyltransferase 1, or for use as a research, diagnostic,or therapeutic agent in accordance with the present invention.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocesssing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons etal., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282,430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir etal., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15,188-200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of antisense strand ofthe duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., Science, 2002, 295, 694-697).

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between diacylglycerol acyltransferase 1 and a disease state,phenotype, or condition. These methods include detecting or modulatingdiacylglycerol acyltransferase 1 comprising contacting a sample, tissue,cell, or organism with the compounds of the present invention, measuringthe nucleic acid or protein level of diacylglycerol acyltransferase 1and/or a related phenotypic or chemical endpoint at some time aftertreatment, and optionally comparing the measured value to a non-treatedsample or sample treated with a further compound of the invention. Thesemethods can also be performed in parallel or in combination with otherexperiments to determine the function of unknown genes for the processof target validation or to determine the validity of a particular geneproduct as a target for treatment or prevention of a particular disease,condition, or phenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention are utilized for diagnostics,therapeutics, prophylaxis and as research reagents and kits. In oneembodiment, such compositions of the invention are useful in the areasof obesity and obesity-associated disorders, such as obesity-relatedinsulin resistance. Furthermore, antisense oligonucleotides, which areable to inhibit gene expression with exquisite specificity, are oftenused by those of ordinary skill to elucidate the function of particulargenes or to distinguish between functions of various members of abiological pathway.

For use in kits and diagnostics, the compounds of the present invention,either alone or in combination with other compounds or therapeutics, areused as tools in differential and/or combinatorial analyses to elucidateexpression patterns of a portion or the entire complement of genesexpressed within cells and tissues.

As used herein the term “system” is defined as any organism, cell, cellculture or tissue that expresses, or is made competent to expressproducts of the gene encoding diacylglycerol acyltransferase 1. Theseinclude, but are not limited to, humans, transgenic animals, cells, cellcultures, tissues, xenografts, transplants and combinations thereof.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundsthat affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encodingdiacylglycerol acyltransferase 1. For example, oligonucleotides that areshown to hybridize with such efficiency and under such conditions asdisclosed herein as to be effective diacylglycerol acyltransferase 1inhibitors will also be effective primers or probes under conditionsfavoring gene amplification or detection, respectively. These primersand probes are useful in methods requiring the specific detection ofnucleic acid molecules encoding diacylglycerol acyltransferase 1 and inthe amplification of said nucleic acid molecules for detection or foruse in further studies of diacylglycerol acyltransferase 1.Hybridization of the antisense oligonucleotides, particularly theprimers and probes, of the invention with a nucleic acid encodingdiacylglycerol acyltransferase 1 can be detected by means known in theart. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of diacylglycerol acyltransferase 1 in a sample may also beprepared.

Among diagnostic uses is the measurement of diacylglycerolacyltransferase 1 levels in patients to identify those who may benefitfrom a treatment strategy aimed at attenuation of inflammation. Suchpatients suitable for diagnosis include patients with obesity, orrelated disorders, including diabetes and cardiac disorders. Suchdisorders may be related to elevated serum glucose levels, elevatedcirculating insulin levels, elevated fasted serum insulin levels,elevated circulating triglycerides, elevated liver triglycerides andelevated free fatty acids in liver.

The specificity and sensitivity of antisense are also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofdiacylglycerol acyltransferase 1 is treated by administering antisensecompounds in accordance with this invention. For example, in onenon-limiting embodiment, the methods comprise the step of administeringto the animal in need of treatment, a therapeutically effective amountof a diacylglycerol acyltransferase 1 inhibitor. The diacylglycerolacyltransferase 1 inhibitors of the present invention effectivelyinhibit the activity of diacylglycerol acyltransferase 1 or inhibit theexpression of diacylglycerol acyltransferase 1. For example, such acompound or composition that reduces levels of diacylglycerolacyltransferase 1 is useful to prevent morbidity and mortality forsubjects with obesity-related disorders. For example, as demonstrated inthe examples, reduction in DGAT1 can result in the reduction of elevatedlevels of serum glucose, circulating insulin, fasted serum, circulatingtriglycerides, liver triglycerides and free fatty acids in liver. Thus,DGAT-1 inhibitors are useful in the treatment of a variety of diabetes,obesity, and cardiac disorders, including acute coronary syndrome. Sucha composition is useful for reducing inflammation mediated bydiacylglycerol acyltransferase 1 in a subject, e.g., to treat or preventor reduce the progression of, atherosclerosis; to treat or prevent orreduce the progression of, acute vascular damage at atheroscleroticplaque sites or in coronary arteries; or to treat or prevent or reducethe progression of, damage caused by inflammation associated withmyocardial infarctions or renal inflammation. Still other therapeutic orprophylactic methods using diacylglycerol acyltransferase 1 inhibitorycompounds of this invention include to treat patients with coronaryartery stenting or liver disorders.

In one embodiment, the activity or expression of diacylglycerolacyltransferase 1 in an animal is inhibited by about 10%. Preferably,the activity or expression of diacylglycerol acyltransferase 1 in ananimal is inhibited by about 30%. More preferably, the activity orexpression of diacylglycerol acyltransferase 1 in an animal is inhibitedby 50% or more. Thus, the oligomeric compounds modulate expression ofdiacylglycerol acyltransferase 1 mRNA by at least 10%, by at least 20%,by at least 25%, by at least 30%, by at least 40%, by at least 50%, byat least 60%, by at least 70%, by at least 75%, by at least 80%, by atleast 85%, by at least 90%, by at least 95%, by at least 98%, by atleast 99%, or by 100%.

For example, the reduction of the expression of diacylglycerolacyltransferase 1 may be measured in serum, adipose tissue, liver or anyother body fluid, tissue or organ of the animal. Preferably, the cellscontained within said fluids, tissues or organs being analyzed contain anucleic acid molecule encoding diacylglycerol acyltransferase 1 and/ordiacylglycerol acyltransferase 1 itself.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

F. Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage, i.e. a singleinverted nucleoside residue that may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleotides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e. the backbone), of the nucleotide units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified Sugars

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

A further preferred modification of the sugar includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in International Patent PublicationNos. WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992,and U.S. Pat. No. 6,287,860, the entire disclosure of which areincorporated herein by reference. Conjugate moieties include but are notlimited to lipid moieties such as a cholesterol moiety, cholic acid, athioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999), which is incorporated herein byreference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds that arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

In one embodiment, desirable chimeric oligonucleotides are 20nucleotides in length, composed of a central region consisting of ten2′-deoxynucleotides, flanked on both sides (5′ and 3′ directions) byfive 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside linkagesare phosphorothioate throughout the oligonucleotide and all cytidineresidues are 5-methylcytidines.

In another embodiment certain preferred chimeric oligonucleotides arethose disclosed in the Examples herein, particularly Example 15.Particularly preferred chimeric oligonucleotides are those referred toas ISIS 191643, ISIS 191647, ISIS 191635, and ISIS 191695.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

G. Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof. Accordingly, for example, the disclosure is alsodrawn to prodrugs and pharmaceutically acceptable salts of the compoundsof the invention, pharmaceutically acceptable salts of such prodrugs,and other bioequivalents. The term “prodrug” indicates a therapeuticagent that is prepared in an inactive form that is converted to anactive form (i.e., drug) within the body or cells thereof by the actionof endogenous enzymes or other chemicals and/or conditions. Inparticular, prodrug versions of the oligonucleotides of the inventionare prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivativesaccording to the methods disclosed in International Patent PublicationNo. WO 93/24510 to Gosselin et al., published Dec. 9, 1993, or inInternational Patent Publication No. WO 94/26764 and U.S. Pat. No.5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes, which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298, filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. Published Patent Application No. 2003/0040497 (Feb. 27,2003) and its parent applications; U.S. Published Patent Application No.2003/0027780 (Feb. 6, 2003) and its parent applications; and U.S. patentapplication Ser. No. 10/071,822, filed Feb. 8, 2002, each of which isincorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents, which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemo-therapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 μgto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence limes and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and InternationalPatent Publication No. WO 02/36743; 5′-O-Dimethoxytrityl-thymidineintermediate for 5-methyl dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,T-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O—[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or 5,366,878, herein incorporated by reference.

Alkylphosphonothioate oligonucleotides are prepared as described inInternational Patent Application Nos. PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 3 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group, which has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine, which notonly cleaves the oligonucleotide from the solid support but also removesthe acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron-withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid.

Example 4 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and 2′deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspectrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Design and Screening of Duplexed Antisense Compounds TargetingDiacylglycerol Acyltransferase 1

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target diacylglycerolacyltransferase 1. The nucleobase sequence of the antisense strand ofthe duplex comprises at least a portion of an oligonucleotide inTable 1. The ends of the strands may be modified by the addition of oneor more natural or modified nucleobases to form an overhang. The sensestrand of the dsRNA is then designed and synthesized as the complementof the antisense strand and may also contain modifications or additionsto either terminus. For example, in one embodiment, both strands of thedsRNA duplex would be complementary over the central nucleobases, eachhaving overhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO: 228) and having a two-nucleobaseoverhang of Deoxythymidine (dT) would have the following structure(Antisense SEQ ID NO: 229, Complement SEQ ID NO: 230):

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 228) may be preparedwith blunt ends (no single stranded overhang) as shown (Antisense SEQ IDNO: 228, Complement SEQ ID NO: 231):

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 uM. Once diluted, 30μL of each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 μL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 uM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate diacylglycerol acyltransferase 1 expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1™ reduced-serum medium(Gibco BRL) and then treated with 130 μL of OPTI-MEM-1™ mediumcontaining 12 μg/mL LIPOFECTIN™ reagent (Gibco BRL) and the desiredduplex antisense compound at a final concentration of 200 nM. After 5hours of treatment, the medium is replaced with fresh medium. Cells areharvested 16 hours after treatment, at which time RNA is isolated andtarget reduction measured by RT-PCR.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full-length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis were determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQapparatus) or, for individually prepared samples, on a commercial CEapparatus (e.g., Beckman P/ACE™ 5000, ABI 270 apparatus). Base andbackbone composition was confirmed by mass analysis of the compoundsutilizing electrospray-mass spectroscopy. All assay test plates werediluted from the master plate using single and multi-channel roboticpipettors. Plates were judged to be acceptable if at least 85% of thecompounds on the plate were at least 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

HepG2 Cells:

The human hepatoblastoma cell line HepG2 was obtained from the AmericanType Culture Collection (Manassas, Va.). HepG2 cells were routinelycultured in Eagle's MEM supplemented with 10% fetal calf serum,non-essential amino acids, and 1 mM sodium pyruvate (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

b.END Cells:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Institute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM supplemented with 10% fetal bovine serum(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 24-well plates (Falcon-Primaria#3047) at a density of 40,000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Primary Rat Hepatocytes:

Primary rat hepatocytes are prepared from Sprague-Dawley rats purchasedfrom Charles River Labs (Wilmington, Mass.) and are routinely culturedin DMEM, high glucose (Invitrogen, Carlsbad, Calif.) supplemented with10% fetal bovine serum (Invitrogen, Carlsbad, Calif.), 100 units per mLpenicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, Calif.).Cells are seeded into 96-well plates (Falcon-Primaria #353872, BDBiosciences, Bedford, Mass.) at a density of 4000-6000 cells/well fortreatment with the oligomeric compounds of the invention.

Treatment with Antisense Compounds:

When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 24-well plates, wells were washedonce with 400 μL Eagle's DMEM and then treated with 100 μL of Eagle'sDMEM containing 3.75 μg/mL LIPOFECTIN™ reagent (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4-7 hoursof treatment at 37° C., the medium was replaced with Eagle's DMEMsupplemented with 10% fetal bovine serum. Cells were harvested 20-24hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of DiacylglycerolAcyltransferase 1 Expression

Antisense modulation of diacylglycerol acyltransferase 1 expression canbe assayed in a variety of ways known in the art. For example,diacylglycerol acyltransferase 1 mRNA levels can be quantitated by,e.g., Northern blot analysis, competitive polymerase chain reaction(PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+ mRNA. The preferred method of RNA analysis of the presentinvention is the use of total cellular RNA as described in otherexamples herein. Methods of RNA isolation are well known in the art.Northern blot analysis is also routine in the art. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of diacylglycerol acyltransferase 1 can be quantitated ina variety of ways well known in the art, such as immunoprecipitation,Western blot analysis (immunoblotting), enzyme-linked immunosorbentassay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodiesdirected to diacylglycerol acyltransferase 1 can be identified andobtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional monoclonal or polyclonal antibody generation methodswell known in the art.

Example 11 Design of Phenotypic Assays and In Vivo Studies for the Useof Diacylglycerol Acyltransferase 1 Inhibitors Phenotypic Assays

Once diacylglycerol acyltransferase 1 inhibitors have been identified bythe methods disclosed herein, the compounds are further investigated inone or more phenotypic assays, each having measurable endpointspredictive of efficacy in the treatment of a particular disease state orcondition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of diacylglycerol acyltransferase 1 in health anddisease. Representative phenotypic assays, which can be purchased fromany one of several commercial vendors, include those for determiningcell viability, cytotoxicity, proliferation or cell survival (MolecularProbes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assaysincluding enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences,Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.),cell regulation, signal transduction, inflammation, oxidative processesand apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated withdiacylglycerol acyltransferase 1 inhibitors identified from the in vitrostudies as well as control compounds at optimal concentrations which aredetermined by the methods described above. At the end of the treatmentperiod, treated and untreated cells are analyzed by one or more methodsspecific for the assay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status, which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the genotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the diacylglycerolacyltransferase 1 inhibitors. Hallmark genes, or those genes suspectedto be associated with a specific disease state, condition, or phenotype,are measured in both treated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

The clinical trial is subjected to rigorous controls to ensure thatindividuals are not unnecessarily put at risk and that they are fullyinformed about their role in the study. To account for the psychologicaleffects of receiving treatments, volunteers are randomly given placeboor diacylglycerol acyltransferase 1 inhibitor. Furthermore, to preventthe doctors from being biased in treatments, they are not informed as towhether the medication they are administering is a diacylglycerolacyltransferase 1 inhibitor or a placebo. Using this randomizationapproach, each volunteer has the same chance of being given either thenew treatment or the placebo.

Volunteers receive either the diacylglycerol acyltransferase 1 inhibitoror placebo for eight week period with biological parameters associatedwith the indicated disease state or condition being measured at thebeginning (baseline measurements before any treatment), end (after thefinal treatment), and at regular intervals during the study period. Suchmeasurements include the levels of nucleic acid molecules encodingdiacylglycerol acyltransferase 1 or the level of diacylglycerolacyltransferase 1 in body fluids, tissues or organs compared topre-treatment levels. Other measurements include, but are not limitedto, indices of the disease state or condition being treated, bodyweight, blood pressure, serum titers of pharmacologic indicators ofdisease or toxicity as well as ADME (absorption, distribution,metabolism and excretion) measurements.

Information recorded for each patient includes age (years), gender,height (cm), family history of disease state or condition (yes/no),motivation rating (some/moderate/great) and number and type of previoustreatment regimens for the indicated disease or condition.

Volunteers taking part in this study are healthy adults (age 18 to 65years) and roughly an equal number of males and females participate inthe study. Volunteers with certain characteristics are equallydistributed for placebo and diacylglycerol acyltransferase 1 inhibitortreatment. In general, the volunteers treated with placebo have littleor no response to treatment, whereas the volunteers treated with thediacylglycerol acyltransferase 1 inhibitor show positive trends in theirdisease state or condition index at the conclusion of the study.

Example 12 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation areroutine in the art. Briefly, for cells grown on 96-well plates, growthmedium was removed from the cells and each well was washed with 200 μLcold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 MNaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added toeach well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 9™ kit and buffers purchased fromQiagen Inc. (Valencia, Calif.) following the manufacturer's recommendedprocedures. Briefly, for cells grown on 96-well plates, growth mediumwas removed from the cells and each well was washed with 200 μL coldPBS. 150 μL Buffer RLT was added to each well and the plate vigorouslyagitated for 20 seconds. 150 μL of 70% ethanol was then added to eachwell and the contents mixed by pipetting three times up and down. Thesamples were then transferred to the RNEASY 96™ well plate attached to aQIAVAC™ manifold fitted with a waste collection tray and attached to avacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 wasadded to each well of the RNEASY 96™ plate and incubated for 15 minutesand the vacuum was again applied for 1 minute. An additional 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumwas applied for 2 minutes. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 90seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 3 minutes.

The plate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 140 μL of RNAse free water into eachwell, incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN® Bio-Robot™ 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of DiacylglycerolAcyltransferase 1 mRNA Levels

Quantitation of diacylglycerol acyltransferase 1 mRNA levels wasaccomplished by real-time quantitative PCR using the ABI PRISM™ 7600,7700, or 7900 Sequence Detection System (PE-Applied Biosystems, FosterCity, Calif.) according to manufacturer's instructions. This is aclosed-tube, non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR in which amplification productsare quantitated after the PCR is completed, products in real-timequantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 5′ end of the probe and a quencherdye (e.g., TAMRA, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end ofthe probe.

When the probe and dyes are intact, reporter dye emission is quenched bythe proximity of the 3′ quencher dye. During amplification, annealing ofthe probe to the target sequence creates a substrate that can be cleavedby the 5′-exonuclease activity of Taq polymerase. During the extensionphase of the PCR amplification cycle, cleavage of the probe by Taqpolymerase releases the reporter dye from the remainder of the probe(and hence from the quencher moiety) and a sequence-specific fluorescentsignal is generated. With each cycle, additional reporter dye moleculesare cleaved from their respective probes, and the fluorescence intensityis monitored at regular intervals by laser optics built into the ABIPRISM™ Sequence Detection System.

In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 30 μL PCR cocktail(2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq enzyme, 5Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 20 μL total RNA solution (20-200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq enzyme, 45 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ reagent(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).Methods of RNA quantification by RiboGreen™ reagent are taught in Jones,L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 180 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 20 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 reader (PE Applied Biosystems) with excitation at485 nm and emission at 530 nm.

Probes and primers to human diacylglycerol acyltransferase 1 weredesigned to hybridize to a human diacylglycerol acyltransferase 1sequence, using published sequence information (GenBank accession numberNM_(—)012079.2, incorporated herein as SEQ ID NO: 4). For humandiacylglycerol acyltransferase 1 the PCR primers were:

forward primer: TCCCCGCATCCGGAA (SEQ ID NO: 5)reverse primer: CTGGGTGAAGAACAGCATCTCA (SEQ ID NO: 6) and the PCR probewas: FAM-CGCTTTCTGCTGCGACGGATCC-TAMRA(SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is thequencher dye. For human GAPDH the PCR primers were:forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 8)reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the PCR probewas: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE isthe fluorescent reporter dye and TAMRA is the quencher dye.

Probes and primers to mouse diacylglycerol acyltransferase 1 weredesigned to hybridize to a mouse diacylglycerol acyltransferase Isequence, using published sequence information (GenBank accession numberAF078752.1, incorporated herein as SEQ ID NO: 11). For mousediacylglycerol acyltransferase 1 the PCR primers were:

forward primer: GTTCCGCCTCTGGGCATT (SEQ ID NO: 12)reverse primer: GAATCGGCCCACAATCCA (SEQ ID NO: 13) and the PCR probewas: FAM-CAGCCATGATGGCTCAGGTCCCACT-TAMRA(SEQ ID NO: 14) where FAM is the fluorescent reporter dye and TAMRA isthe quencher dye.

For mouse GAPDH the PCR primers were:

forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 15)reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 16) and the PCR probewas: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 17) whereJOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14 Northern Blot Analysis of Diacylglycerol Acyltransferase 1mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ reagent (TEL-TEST “B”Inc., Friendswood, Tex.). Total RNA was prepared followingmanufacturer's recommended protocols. Twenty micrograms of total RNA wasfractionated by electrophoresis through 1.2% agarose gels containing1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon,Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes(Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillarytransfer using a Northern/Southern Transfer buffer system (TEL-TEST “B”Inc., Friendswood, Tex.). RNA transfer was confirmed by UVvisualization. Membranes were fixed by UV cross-linking using aSTRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.)and then probed using QUICKHYB™ hybridization solution (Stratagene, LaJolla, Calif.) using manufacturer's recommendations for stringentconditions.

To detect human diacylglycerol acyltransferase 1, a human diacylglycerolacyltransferase 1 specific probe was prepared by PCR using the forwardprimer TCCCCGCATCCGGAA (SEQ ID NO: 5) and the reverse primerCTGGGTGAAGAACAGCATCTCA (SEQ ID NO: 6). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forhuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

To detect mouse diacylglycerol acyltransferase 1, a mouse diacylglycerolacyltransferase 1 specific probe was prepared by PCR using the forwardprimer GTTCCGCCTCTGGGCATT (SEQ ID NO: 12) and the reverse primerGAATCGGCCCACAATCCA (SEQ ID NO: 13). To normalize for variations inloading and transfer efficiency membranes were stripped and probed formouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ apparatus and IMAGEQUANT™ Software V3.3 (MolecularDynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels inuntreated controls.

Example 15 Antisense Inhibition of Human Diacylglycerol Acyltransferase1 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the humandiacylglycerol acyltransferase 1 RNA, using published sequences (GenBankaccession number NM_(—)012079.2, incorporated herein as SEQ ID NO: 4).The compounds are shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe compound binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humandiacylglycerol acyltransferase 1 mRNA levels by quantitative real-timePCR as described in other examples herein. Data are averages from threeexperiments in which HepG2 cells were treated with 75 nM of theantisense oligonucleotides of the present invention. The positivecontrol for each data point is identified in the table by sequence IDnumber. If present, “N.D.” indicates “no data”.

TABLE 1Inhibition of human diacylglycerol acyltransferase 1 mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET CONTROL SEQ ID TARGET % SEQ SEQ ID ISIS # REGION NO SITE SEQUENCEINHIB ID NO NO 191617 5′UTR 4 1 gccgcctctctcgtccattc 57 18 1 1916195′UTR 4 21 gagccgctaactaatggacg 37 19 1 191621 5′UTR 4 41acaacggctgcgttgctccg 30 20 1 191623 5′UTR 4 71 ccgcccgcgtcaggcccgtc 4021 1 191625 5′UTR 4 91 gcctcaccagcgcgttcaac 20 22 1 191627 5′UTR 4 120ccctgccggccgccgtagcc 24 23 1 191629 5′UTR 4 151 ctccgggccctagacaacgg 4524 1 191631 5′UTR 4 181 gttcgtagcgcccgaggcgc 53 25 1 191633 5′UTR 4 211cccggccgcagccaagcgtg 44 26 1 191635 Start Codon 4 231gcccatggcctcagcccgca 77 27 1 191637 Coding 4 281 tggctcgagggccgcgaccc 5828 1 191639 Coding 4 301 ccgcaggcccgccgccgccg 49 29 1 191641 Coding 4321 ccgcacctcttcttccgccg 40 30 1 191643 Coding 4 401acgccggcgtctccgtcctt 92 31 1 191645 Coding 4 421 gctcccagtggccgctgccc 6032 1 191647 Coding 4 441 ctgcaggcgatggcacctca 85 33 1 191649 Coding 4491 aggatgccacggtagttgct 62 34 1 191651 Coding 4 511gcatcaccacacaccagttc 37 35 1 191653 Coding 4 561 gccatacttgatgaggttct 4836 1 191655 Coding 4 651 gacattggccgcaataacca 47 37 1 191657 Coding 4681 cttctcaacctggaatgcag 29 38 1 191659 Coding 4 721gcagtcccgcctgctccgtc 50 39 1 191661 Coding 4 741 caggttggctacgtgcagca 3140 1 191663 Coding 4 781 ccagtaagaccacagccgct 62 41 1 191665 Coding 4831 ggtgtgcgccatcagcgcca 59 42 1 191667 Coding 4 931cagcactgctggccttcttc 52 43 1 191669 Coding 4 1021 tgagctcgtagcacaaggtg43 44 1 191671 Coding 4 1121 cactgctggatcagccccac 20 45 1 191673 Coding4 1181 atgcgtgagtagtccatgtc 59 46 1 191675 Coding 4 1231tgagccagatgaggtgattg 62 47 1 191677 Coding 4 1281 gagctcagccacggcattca76 48 1 191679 Coding 4 1351 tctgccagaagtaggtgaca 30 49 1 191681 Coding4 1611 gatgagcgacagccacacag 21 50 1 191683 Coding 4 1671ctcatagttgagcacgtagt 73 51 1 191685 3′UTR 4 1721 cagtgagaagccaggccctc 6852 1 191687 3′UTR 4 1781 ccatccccagcactcgaggc 68 53 1 191689 3′UTR 41801 aggatgctgtgcagccaggc 73 54 1 191691 3′UTR 4 1851ggtgcaggacagagccccat 72 55 1 191693 3′UTR 4 1881 gtgtctggcctgctgtcgcc 7156 1 191695 3′UTR 4 1901 ctcccagctggcatcagact 76 57 1

As shown in Table 1, SEQ ID Nos. 18, 25, 27, 28, 31, 32, 33, 34, 39, 41,42, 43, 46, 47, 48, 51, 52, 53, 54, 55, 56 and 57 demonstrated at least50% inhibition of human diacylglycerol acyltransferase 1 expression inthis assay and are therefore preferred. More preferred are SEQ ID NOs31, 33, 27, and 57. The target regions to which these preferredsequences are complementary are herein referred to as “preferred targetsegments” and are therefore preferred for targeting by compounds of thepresent invention. These preferred target segments are shown in Table 3.The sequences represent the reverse complement of the preferredantisense compounds shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number on the particular target nucleic acid towhich the oligonucleotide binds. Also shown in Table 3 is the species inwhich each of the preferred target segments was found.

Example 16 Antisense Inhibition of Mouse Diacylglycerol Acyltransferase1 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a second series of antisensecompounds was designed to target different regions of the mousediacylglycerol acyltransferase 1 RNA, using published sequences (GenBankaccession number AF078752.1, incorporated herein as SEQ ID NO: 11). Thecompounds are shown in Table 2. “Target site” indicates the first(5′-most) nucleotide number on the particular target nucleic acid towhich the compound binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mousediacylglycerol acyltransferase 1 mRNA levels by quantitative real-timePCR as described in other examples herein. Data are averages from threeexperiments in which b.END cells were treated with the antisenseoligonucleotides of the present invention. If present, “N.D.” indicates“no data”.

TABLE 2Inhibition of mouse diacylglycerol acyltransferase 1 mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET SEQ ID TARGET % SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB NO191723 5′UTR 11 1 ctacttatttccattcatcc 2 58 191724 5′UTR 11 21tatcctaagtatgcctaatt 0 59 191725 5′UTR 11 31 gcttgagccctatcctaagt 0 60191726 5′UTR 11 61 ctcgtcgcggcccaatcttc 21 61 191727 Start Codon 11 81cccatggcttcggcccgcac 48 62 191729 Coding 11 191 cagccgcgtctcgcacctcg 7463 191730 Coding 11 232 cggagccggcgcgtcacccc 63 64 191731 Coding 11 281ccacgctggtccgcccgtct 67 65 191732 Coding 11 301 cagatcccagtagccgtcgc 5966 191733 Coding 11 321 tcttgcagacgatggcacct 49 67 191734 Coding 11 371tcaggataccacgataattg 48 68 191735 Coding 11 391 cagcatcaccacacaccaat 5269 191736 Coding 11 411 aaccttgcattactcaggat 62 70 191737 Coding 11 451atccaccaggatgccatact 29 71 191738 Coding 11 471 agagacaccacctggatagg 4272 191740 Coding 11 601 cagcagccccatctgctctg 63 73 191741 Coding 11 621gccaggttaaccacatgtag 58 74 191742 Coding 11 661 aaccagtaaggccacagctg 1675 191743 Coding 11 681 cccactggagtgatagactc 42 76 191744 Coding 11 711atggagtatgatgccagagc 53 77 191745 Coding 11 771 acccttcgctggcggcacca 6878 191746 Coding 11 841 tggatagctcacagcttgct 56 79 191747 Coding 11 861tctcggtaggtcaggttgtc 32 80 191748 Coding 11 961 ctcaagaactcgtcgtagca 6081 191749 Coding 11 1001 gttggatcagccccacttga 37 82 191750 Coding 111061 gtgaatagtccatatccttg 48 83 191751 Coding 11 1081taagagacgctcaatgatcc 18 84 191752 Coding 11 1161 tctgccacagcattgagaca 5085 191753 Coding 11 1201 ccaatctctgtagaactcgc 55 86 191754 Coding 111221 gtgacagactcagcattcca 56 87 191755 Coding 11 1271gtctgatgcaccacttgtgc 72 88 191756 Coding 11 1301 tgccatgtctgagcataggc 7089 191757 Coding 11 1331 atactcctgtcctggccacc 65 90 191759 Coding 111471 attgccatagttcccttgga 68 91 191760 Coding 11 1491agtgtcacccacacagctgc 66 92 191761 Coding 11 1511 ccaccggttgcccaatgatg 7193 191762 Coding 11 1531 gtggacatacatgagcacag 62 94 191763 Coding 111551 tagttgagcacgtagtagtc 40 95 191764 Stop Codon 11 1586ctttggcagtagctcatacc 37 96 191765 3′UTR 11 1621 tccagaactccaggcccagg 5997

As shown in Table 2, SEQ ID Nos. 63, 64, 65, 66, 69, 70, 73, 74, 77, 78,79, 81, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 and 97 demonstrated atleast 50% inhibition of mouse diacylglycerol acyltransferase 1expression in this experiment and are therefore preferred. Morepreferred are SEQ ID Nos. 63, 88, 91, and 93. The target regions towhich these preferred sequences are complementary are herein referred toas “preferred target segments” and are therefore preferred for targetingby compounds of the present invention. These preferred target segmentsof the mRNA are shown in Table 3 as the appropriate RNA sequence, wherethymine (T) has been replaced with uracil (U) to reflect correctrepresentation of an RNA sequence. The sequences represent the reversecomplement of the preferred antisense compounds shown in Tables 1 and 2.“Target site” indicates the first (5′-most) nucleotide number on theparticular target nucleic acid to which the oligonucleotide binds. Alsoshown in Table 3 is the species in which each of the preferred targetsegments was found.

TABLE 3Sequence and position of preferred target segments identified in diacylglycerolacyltransferase 1. TARGET SITE SEQ ID TARGET REV COMP SEQ ID ID NO SITESEQUENCE OF SEQ ID ACTIVE IN NO 108088 4 1 gaauggacgagagaggcggc 18H. sapiens 98 108094 4 151 ccguugucuagggcccggag 24 H. sapiens 99 1080954 181 gcgccucgggcgcuacgaac 25 H. sapiens 100 108096 4 211cacgcuuggcugcggccggg 26 H. sapiens 101 108097 4 231 ugcgggcugaggccaugggc27 H. sapiens 102 108098 4 281 gggucgcggcccucgagcca 28 H. sapiens 103108099 4 301 cggcggcggcgggccugcgg 29 H. sapiens 104 108101 4 401aaggacggagacgccggcgu 31 H. sapiens 105 108102 4 421 gggcagcggccacugggagc32 H. sapiens 106 108103 4 441 ugaggugccaucgccugcag 33 H. sapiens 107108104 4 491 agcaacuaccguggcauccu 34 H. sapiens 108 108106 4 561agaaccucaucaaguauggc 36 H. sapiens 109 108107 4 651 ugguuauugcggccaauguc37 H. sapiens 110 108109 4 721 gacggagcaggcgggacugc 39 H. sapiens 111108111 4 781 agcggcuguggucuuacugg 41 H. sapiens 112 108112 4 831uggcgcugauggcgcacacc 42 H. sapiens 113 108113 4 931 gaagaaggccagcagugcug43 H. sapiens 114 108114 4 1021 caccuugugcuacgagcuca 44 H. sapiens 115108116 4 1181 gacauggacuacucacgcau 46 H. sapiens 116 108117 4 1231caaucaccucaucuggcuca 47 H. sapiens 117 108118 4 1281ugaaugccguggcugagcuc 48 H. sapiens 118 108121 4 1671acuacgugcucaacuaugag 51 H. sapiens 119 108122 4 1721gagggccuggcuucucacug 52 H. sapiens 120 108123 4 1781gccucgagugcuggggaugg 53 H. sapiens 121 108124 4 1801gccuggcugcacagcauccu 54 H. sapiens 122 108125 4 1851auggggcucuguccugcacc 55 H. sapiens 123 108126 4 1881ggcgacagcaggccagacac 56 H. sapiens 124 108127 4 1901agucugaugccagcugggag 57 H. sapiens 125 108139 11 81 gugcgggccgaagccauggg62 M. musculus 126 108141 11 191 cgaggugcgagacgcggcug 63 M. musculus 127108142 11 232 ggggugacgcgccggcuccg 64 M. musculus 128 108143 11 281agacgggcggaccagcgugg 65 M. musculus 129 108144 11 301gcgacggcuacugggaucug 66 M. musculus 130 108145 11 321aggugccaucgucugcaaga 67 M. musculus 131 108146 11 371caauuaucgugguauccuga 68 M. musculus 132 108147 11 391auugguguguggugaugcug 69 M. musculus 133 108148 11 411auccugaguaaugcaagguu 70 M. musculus 134 108152 11 601cagagcagauggggcugcug 73 M. musculus 135 108153 11 621cuacaugugguuaaccuggc 74 M. musculus 136 108156 11 711gcucuggcaucauacuccau 77 M. musculus 137 108157 11 771uggugccgccagcgaagggu 78 M. musculus 138 108158 11 841agcaagcugugagcuaucca 79 M. musculus 139 108160 11 961ugcuacgacgaguucuugag 81 M. musculus 140 108162 11 1061caaggauauggacuauucac 83 M. musculus 141 108164 11 1161ugucucaaugcuguggcaga 85 M. musculus 142 108165 11 1201gcgaguucuacagagauugg 86 M. musculus 143 108166 11 1221uggaaugcugagucugucac 87 M. musculus 144 108167 11 1271gcacaaguggugcaucagac 88 M. musculus 145 108168 11 1301gccuaugcucagacauggca 89 M. musculus 146 108169 11 1331gguggccaggacaggaguau 90 M. musculus 147 108171 11 1471uccaagggaacuauggcaau 91 M. musculus 148 108172 11 1491gcagcugugugggugacacu 92 M. musculus 149 108173 11 1511caucauugggcaaccggugg 93 M. musculus 150 108174 11 1531cugugcucauguauguccac 94 M. musculus 151 108177 11 1621ccugggccuggaguucugga 97 M. musculus 152

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of diacylglycerolacyltransferase 1.

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds, that hybridize toat least a portion of the target nucleic acid.

Example 17 Western Blot Analysis of Diacylglycerol Acyltransferase 1Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μL/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to diacylglycerolacyltransferase 1 is used, with a radiolabeled or fluorescently labeledsecondary antibody directed against the primary antibody species. Bandsare visualized using a PHOSPHORIMAGER™ instrument (Molecular Dynamics,Sunnyvale Calif.).

Example 18 Antisense Inhibition of Mouse Diacylglycerol Acyltransferase1 Expression: Dose Response in b.END Cells

In accordance with the present invention, six oligonucleotides targetedto mouse diacylglycerol acyltransferase 1, ISIS 191729 (SEQ ID NO: 63),ISIS 191731 (SEQ ID NO: 65), ISIS 191755 (SEQ ID NO: 88), ISIS 191756(SEQ ID NO: 89), ISIS 191759 (SEQ ID NO: 91), and ISIS 191761 (SEQ IDNO: 93), were further investigated in a dose response study.

In the dose-response experiment, with mRNA levels as the endpoint, b.ENDcells were treated with ISIS 191729, ISIS 191731, ISIS 191755, ISIS191756, ISIS 191759, or ISIS 191761 at doses of 1, 5, 10, 25, 50, and100 nM oligonucleotide. Data were obtained by real-time quantitative PCRas described in other examples herein and are averaged from threeexperiments and are normalized to untreated control cells. The data areshown in Table 4.

TABLE 4 Inhibition of mouse diacylglycerol acyltransferase 1 mRNA levelsby chimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap: dose response Dose (nM) SEQ ID 1 5 10 25 50 100 ISIS # NO %Inhibition 191729 63 26 62 78 80 83 83 191731 65 27 58 57 58 82 85191755 88 41 59 72 75 83 79 191756 89 13 39 59 65 81 75 191759 91 26 4474 80 82 86 191761 93 23 63 71 80 85 87

From these data, it is evident that all of the oligonucleotidespresented in Table 4 are capable of reducing diacylglycerolacyltransferase 1 mRNA levels in a dose-dependent manner.

Example 19 Effects of Antisense Inhibition of DiacylglycerolAcyltransferase 1 (ISIS 191729 and ISIS 191755) on Serum GlucoseLevels—In Vivo Studies

Leptin is a hormone produced by fat cells that regulates appetite.Deficiencies in this hormone in both humans and non-human animals leadsto obesity. ob/ob mice have a mutation in the leptin gene which resultsin obesity and hyperglycemia. As such, these mice are a useful model forthe investigation of obesity and diabetes and treatments designed totreat these conditions. ob/ob mice have higher circulating levels ofinsulin and are less hyperglycemic than db/db mice, which harbor amutation in the leptin receptor. In accordance with the presentinvention, the oligomeric compounds of the invention are tested in theob/ob model of obesity and diabetes as potential agents to lower serumglucose levels.

Seven-week old male C57Bl/6J-Lep ob/ob mice (Jackson Laboratory, BarHarbor, Me.) were fed a Purina 5015 diet (10-15% fat) and were evaluatedover the course of 4 weeks for the effects of ISIS 191729 (SEQ ID No:63) and ISIS 191755 (SEQ ID NO: 88) on serum glucose levels. Mice weredosed intraperitoneally twice a week with 25 mg/kg ISIS 191729 or ISIS191755. Control animals received saline treatment twice per week for 4weeks. Each group consisted of 8 animals. At the end of the treatmentperiod, animals were sacrificed. Glucose levels in serum were measuredusing a YSI glucose analyzer (YSI Scientific, Yellow Springs, Ohio).Data are presented as the average of the 8 animals in each treatmentgroup.

Both antisense oligonucleotides were able to reduce serum glucose levelsrelative to the saline-treated animals. Before any treatment was started(week 0), the measured glucose levels for each group of animals were357, 368, and 346 mg/dL for the groups that would be treated withsaline, ISIS 191729, and ISIS 191755, respectively. After two weeks,serum glucose levels were 300 and 278 mg/dL for ISIS 191729 and ISIS191755, respectively, compared to 360 mg/dL for saline control. Afterfour weeks of treatment, the serum glucose levels were further reducedto 224 and 188 mg/dL for ISIS 191729 and ISIS 191755, respectively,compared to 313 mg/dL for saline control.

These data indicate that ISIS 191729 and ISIS 191755 significantlyreduced serum glucose levels in vivo. ISIS 191755 also caused no changein food intake or body weight, but reduced epididymal fat pad weight by12%. (See Table 5 for a summary of in vivo data).

Example 20 Effects of Antisense Inhibition of DiacylglycerolAcyltransferase 1 (ISIS 191729 and ISIS 191755) on DiacylglycerolAcyltransferase 1 mRNA Levels in C57BL/6 Mice

In a further embodiment, the mice described in Example 19 were evaluatedfor diacylglycerol acyltransferase 1 expression in liver and whiteadipose tissue. Following the four week treatment with twice weeklyinjections of 25 mg/kg ISIS 191729 (SEQ ID No: 63) and ISIS 191755 (SEQID NO: 88), the mice were sacrificed and liver and white adipose tissueswere procured. Diacylglycerol acyltransferase 1 mRNA levels werequantitated by real-time PCR as described in other examples herein. Datapresented are the average of the 8 animals in each treatment group.

The diacylglycerol acyltransferase 1 mRNA levels in white adipose tissueof the mice dosed with ISIS 191729 were 29% that of the saline-treatedmice, and those dosed with ISIS 191755 were 16% that of thesaline-treated mice. The diacylglycerol acyltransferase 1 mRNA levels inliver of the mice dosed with ISIS 191729 were 8% that of thesaline-treated mice, and those dosed with ISIS 191755 were 4% that ofthe saline-treated mice.

It has been reported in the art that diacylglycerol acyltransferase 1knockout mice demonstrate enhanced resistance to diet-induced obesityand this was not coupled with changes in energy expenditure or plasmaglucose levels in ob/ob mice due to a compensatory upregulation ofdiacylglycerol acyltransferase 2 expression in white adipose tissue. Theresults of studies described herein using antisense compounds totransiently modulate diacylglycerol acyltransferase 1 mRNA levels are incontrast to those seen in the diacylglycerol acyltransferase 1 knockoutstudies. The results shown herein indicate that antisenseoligonucleotides ISIS 191729 and ISIS 191755 are able to reducediacylglycerol acyltransferase 1 mRNA levels, reduce serum glucoselevels, and reduce fat pad weight while not affecting food intake andtotal body weight. (See Table 5 for a summary of in vivo data).

TABLE 5 Effects of ISIS 191729 or ISIS 191755 treatment on serum glucoselevels and diacylglycerol acyltransferase 1 mRNA levels in ob/ob mice.Treated with Biological Marker ISIS ISIS Measured Saline 191729 191755Week Glucose 0 357 368 346 mg/dL 2 360 300 278 4 313 224 188 Tissue mRNALiver 100 8 4 % of control White adipose 100 29 16

Example 21 Antisense Inhibition of Diacylglycerol Acyltransferase 1 in aMouse Model of Diet-Induced Obesity: a 7 Week Study

The C57BL/6 mouse strain is reported to be susceptible tohyperlipidemia-induced atherosclerotic plaque formation. Consequently,when these mice are fed a high-fat diet, they develop diet-inducedobesity. Accordingly these mice are a useful model for the investigationof obesity and treatments designed to treat this condition. In thisstudy, animals with diet-induced obesity were evaluated fordiacylglycerol acyltransferase 1 mRNA expression, circulating plasmaglucose and insulin levels, serum transaminase levels, food intake, bodyweight and metabolic rate.

Male C57BL/6 mice (7-weeks old) received a 60% fat diet for 8 weeks,after which mice were subcutaneously injected twice weekly with a 25mg/kg dose of ISIS 191729 (SEQ ID NO: 63) or ISIS 141923(CCTTCCCTGAAGGTTCCTCC; incorporated herein as SEQ ID NO: 153) for aperiod of 7 weeks. Control animals received subcutaneous salineinjections twice weekly for 7 weeks. Each treatment group consisted of 8mice.

ISIS 141923, a scrambled control oligonucleotide, is a chimericoligonucleotides (“gapmer”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

At study termination, 72 hours after the final injections, the animalswere sacrificed and evaluated for diacylglycerol acyltransferase 1 mRNAexpression in liver and white adipose tissue (WAT). RNA was isolatedfrom liver and mRNA was quantitated as described herein. Diacylglycerolacyltransferase 1 mRNA levels from each treatment group (n=8) wereaveraged. Relative to saline-treated animals, treatment with ISIS 191729resulted in an 86% and 77% reduction in diacylglycerol acyltransferase 1mRNA levels in liver and WAT, respectively. Treatment with the scrambledcontrol ISIS 141923 resulted in 9% decrease and a 23% increase indiacylglycerol acyltransferase 1 mRNA levels in liver and WAT,respectively. These results demonstrate that antisense compoundstargeted to diacylglycerol acyltransferase 1 significantly reduceddiacylglycerol acyltransferase 1 mRNA expression in liver and WAT in amodel of diet-induced obesity.

Example 22 Effects of Antisense Inhibition of DiacylglycerolAcyltransferase 1 in a Mouse Model of Diet-Induced Obesity: CirculatingGlucose and Insulin Levels

In a further embodiment, the mice described in Example 21 were evaluatedfor circulating glucose and insulin levels over the course of treatmentwith saline, ISIS 191729 (SEQ ID NO: (63) and ISIS 141923 (SEQ ID NO:153). Measurements were taken prior to the beginning of the treatmentperiod (week 0), and after 3 and 7 weeks of treatment. Glucose levels inserum were measured using a YSI glucose analyzer (YSI Scientific, YellowSprings, Ohio) and insulin levels in serum were measure using an ratinsulin-specific ELISA kit (ALPCO Diagnostics, Windham, N.H.). Thelevels of circulating glucose (mg/dL) and insulin (ng/mL) levels at theindicated time points are presented in Table 6 as the average resultfrom each treatment group (n=8).

TABLE 6 Effects of antisense inhibition of diacylglycerolacyltransferase 1 on circulating glucose and insulin levels in a mousemodel of diet-induced obesity Biological Weeks of treatment MarkerMeasured Treatment 0 3 7 Glucose levels Saline 223 203 252 mg/dL ISIS141923 196 199 230 ISIS 191729 240 183 206 Insulin levels Saline 1.2 1.81.8 ng/mL ISIS 141923 0.9 1.9 2.2 ISIS 191729 1.5 1.2 0.8

These results illustrate that inhibition of diacylglycerolacyltransferase 1 expression significantly decreased circulating insulinlevels with respect to animals injected with ISIS 141923 or saline.

Example 23 Effects of Antisense Inhibition of DiacylglycerolAcyltransferase 1 in a Mouse Model of Diet-Induced Obesity: SerumTransaminase Levels

In a further embodiment, the mice described in Example 21 were evaluatedfor levels of the serum transaminases ALT and AST. Increases in theseenzymes can indicate hepatotoxicity.

At study termination, seventy-two hours after the final injections, theanimals treated with ISIS 191723 (SEQ ID NO: 63), ISIS 141923 (SEQ IDNO: 153) and saline were sacrificed and evaluated for ALT and AST levelsin serum. ALT and AST were measured by routine methods at LabCorpclinical laboratories (San Diego, Calif.). These results are presentedin Table 7 as the average result from each treatment group (n=8), ininternational units/L (IU/L).

TABLE 7 Effects of antisense inhibition of diacylglycerolacyltransferase 1 on serum transaminase in a mouse model of diet-inducedobesity Serum Transaminases IU/L Treatment AST ALT Saline 84 51 ISIS141923 89 43 ISIS 191729 179 132

In this animal model, hepatotoxicity is defined as an ALT or AST levelabove twice that of the control. These results demonstrate that ISIS191729 resulted in an elevation of ALT and AST levels to approximatelytwice the level of the control.

Example 24 Effects of Antisense Inhibition of DiacylglycerolAcyltransferase 1 in a Mouse Model of Diet-Induced Obesity: Body Weight,Food Intake and Metabolic Rate

In a further embodiment, the mice described in Example 21 were evaluatedfor body weight, food intake and metabolic rate during the course oftreatment with ISIS 191729 (SEQ ID NO: 63), ISIS 141923 (SEQ ID NO: 153)and saline. Body weight and food intake were recorded weekly throughoutthe 7 week study period, and were found not to change significantlyamong the 3 treatment groups. At week 4, metabolic rates were measuredby placing the mice a metabolic chamber for indirect calorimetrymeasurements (Oxymax; Columbus Instruments International, Columbus,Ohio) for a period of 24 hours. Relative to saline- or ISIS141923-treated mice, no significant differences were found in themetabolic rates of mice treated with ISIS 191729. These resultsdemonstrate that treatment with antisense compounds targeted todiacylglycerol acyltransferase 1 did not significantly affect bodyweight, food intake or metabolic rate in a mouse model of diet-inducedobesity.

Example 25 Antisense Inhibition of Diacylglycerol Acyltransferase 1 mRNAin a Mouse Model of Diet-Induced Obesity: a 5 Week Study

The C57BL/6 mouse strain is reported to be susceptible tohyperlipidemia-induced atherosclerotic plaque formation. Consequently,when these mice are fed a high-fat diet, they develop diet-inducedobesity. Accordingly these mice are a useful model for the investigationof obesity and treatments designed to treat this conditions. A 5 weekstudy was performed to further investigate the effects of antisenseinhibition of diacylglycerol acyltransferase 1 in a mouse model ofdiet-induced obesity. In this study, mice receiving treatment withantisense compounds targeted to diacylglycerol acyltransferase 1 wereevaluated for target mRNA expression, tolerance to glucose and insulinchallenges, fasting serum insulin levels, serum free fatty acids, serumtriglycerides and liver tissue triglycerides.

Male C57BL/6 mice (7-weeks old) received a 60% fat diet for 5 weeks,after which mice were subcutaneously injected twice weekly with a 25mg/kg dose of ISIS 141923 (SEQ ID NO: 153) or ISIS 191729 (SEQ ID NO:63) for a period of 5 weeks. Control animals received subcutaneoussaline injections twice per week for 5 weeks. Each treatment groupconsisted of 8 mice.

At study termination, 72 hours after the final injections, the animalswere sacrificed and evaluated for diacylglycerol acyltransferase 1 mRNAexpression in liver and white adipose tissue (WAT). RNA was isolatedfrom liver and WAT and mRNA was quantitated as described herein.Diacylglycerol acyltransferase 1 mRNA levels from each treatment group(n=8) were averaged. Relative to saline-treated animals, treatment withISIS 191729 resulted in a 90% and 82% reduction in diacylglycerolacyltransferase 1 mRNA levels in liver and WAT, respectively. The micetreated with the scrambled control oligonucleotide ISIS 141923 exhibiteda 16% increase and 25% reduction in diacylglycerol acyltransferase 1mRNA levels in liver and WAT, respectively. These results demonstratethat, as was observed in the 7 week study in a mouse model ofdiet-induced obesity, ISIS 191729 significantly reduced diacylglycerolacyltransferase 1 mRNA expression in liver and WAT.

Example 26 Effects of Antisense Inhibition of DiacylglycerolAcyltransferase 1 on Serum Insulin Levels Following a 4 Hour Fast

In a further embodiment, after 4 weeks of treatment with saline, ISIS191729 (SEQ ID NO: 63) or ISIS 141923 (SEQ ID NO: 153) the micedescribed in Example 25 were evaluated for serum insulin levelsfollowing a 4 hour fast. Serum insulin levels were measured using aninsulin-specific ELISA kit (American Laboratory Products, Windham,N.H.). Mice treated with ISIS 191729 had lower fasting serum insulinlevels (0.52 ng/ml) when compared to saline-treated (1.11 ng/ml) or ISIS141923-treated (0.95 ng/ml) mice. Results presented are the averageresult from each treatment group (n=8). These results demonstrate thatantisense compounds targeted to diacylglycerol acyltransferase 1significantly decreased fasting serum insulin levels in a mouse model ofdiet-induced obesity.

Example 27 Effects of Antisense Inhibition of DiacylglycerolAcyltransferase 1 in a Mouse Model of Diet-Induced Obesity: Insulin andGlucose Tolerance Tests

In a further embodiment, the mice described in Example 25 were evaluatedon their performance on insulin and glucose tolerance tests. Throughmeasurement of glucose levels following the injection of a bolus ofglucose or insulin, tolerance tests assess the physiological response toan insulin or glucose challenge.

Glucose tolerance tests (GTT) were performed after 5 weeks of treatmentwith saline, ISIS 141923 (SEQ ID NO: 153) ISIS 191729 (SEQ ID NO: 63).To provide a baseline glucose level, blood glucose levels were measuredbefore the glucose challenge. Mice received intraperitoneal injectionsof glucose at a dose of 1 g/kg. Blood glucose levels were measured 15,30, 60, 90 and 120 minutes post-challenge using an Olympus ClinicalAnalyzer (Olympus America, Melville, N.H.).

Insulin tolerance tests (ITT) were performed after 4 weeks of treatmentwith saline, ISIS 141923 or ISIS 191729. To provide a baseline insulinlevel, blood insulin levels were measured before the glucose challenge.Mice received intraperitoneal injections of insulin at a dose of 0.5units/kg. Blood glucose levels were measured 15, 30, 60, 90 and 120minutes post-challenge using an Olympus Clinical Analyzer (OlympusAmerica, Melville, N.H.).

The results are presented in Table 8 as the average result from eachtreatment group (n=8). Saline-treated mice served as the control towhich insulin and glucose levels were compared.

TABLE 8 Effects of antisense inhibition of diacylglycerolacyltransferase 1 on insulin and glucose tolerance tests Glucose, mg/dLat intervals after challenge 0 15 30 60 90 120 Treatment min min min minmin min Insulin Saline 178 184 131 140 188 217 Tolerance ISIS 141923 171168 121 133 177 213 Test ISIS 191729 160 148 114 113 140 160 GlucoseSaline 103 319 345 292 252 220 Tolerance ISIS 141923 113 313 345 276 211193 Test ISIS 191729 94 267 304 235 187 164

These results demonstrate that in animals treated with ISIS 191729, butnot those treated with saline or ISIS 141923, glucose levels returned toand did not exceed baseline values within two hours following theinsulin challenge. For both insulin and glucose tolerance tests, a graphof the data presented in Table 8 demonstrates an improved area under thecurve following treatment with ISIS 191729, revealing an improvedperformance on glucose and insulin tolerance tests.

Example 28 Effects of Antisense Inhibition of DiacylglycerolAcyltransferase 1 in a Mouse Model of Diet-Induced Obesity: SerumTriglyceride, Liver Triglyceride, and Free Fatty Acid Levels

In a further embodiment, the mice described in Example 25 were evaluatedfor the effects of saline, ISIS 141923 (SEQ ID NO: 153) and ISIS 191729(SEQ ID NO: 63) treatment on serum triglyceride, liver triglyceride, andfree fatty acid levels.

Animals were subjected to a 4 hour fast after the 5 week treatmentperiod for measurement of serum free fatty acids. Serum free fatty acids(4 hour fasting, mEq/L) were measured using a kit for non-esterifiedfree fatty acids (Wako Chemicals, Richmond, Va.). Data are presented inTable 9 as the average of 8 animals in a treatment group.

Following sacrifice of the animals, 72 hours after the last injection,serum was collected and serum triglycerides (mg/dl) were measured byroutine methods at LabCorp clinical laboratories (San Diego, Calif.).Liver tissue was procured and liver triglyceride (mg/g wet tissue)levels were measured using a Triglyceride GPO Assay from RocheDiagnostics (Indianapolis, Ind.). Liver triglyceride levels are used toassess hepatic steatosis, or clearing of lipids from the liver. Theseresults are presented in Table 9 as the average result from 8 animals ina treatment group.

TABLE 9 Effects of antisense inhibition of diacylglycerolacyltransferase 1 on serum triglyceride, liver triglyceride, and freefatty acid levels Biological Marker Measured Free Fatty AcidsTriglycerides Treatment Serum mEq/L Serum mg/dL Liver mg/g Saline 0.7485 71 ISIS 141923 0.68 80 58 ISIS 191729 0.56 56 64

These results demonstrate that antisense compounds targeted todiacylglycerol acyltransferase 1 reduced circulating triglyceride levelsin a mouse model of diet-induced obesity. The data are also suggestiveof a drop in liver triglyceride and free fatty acid levels in a mousemodel of diet-induced obesity.

The data obtained in a mouse model of diet-induced obesity and presentedherein demonstrate that treatment with ISIS 191729 significantlydecreased in diacylglycerol acyltransferase 1 mRNA expression in bothliver and white adipose tissue. The inhibition of target mRNA expressionresulted in a reduction in circulating insulin and triglyceride levelsand an improved area under the curve following glucose and insulintolerance tests. The data also suggest a reduction in circulating freefatty acids and liver triglycerides. These effects were independent ofchanges in food intake, metabolic rate or body weight. Together, thesedata reveal that ISIS 191729 treatment improved the overall metabolicrate and cardiovascular risk profile in a model of diet-induced obesity.Moreover, the effects of a transient reduction in diacylglycerolacyltransferase 1 expression, described herein, were in contrast tofindings in the diacylglycerol transferase 1 mouse gene disruptionmodel, where said mice are resistant to diet-induced obesity andprotected against insulin resistance.

Example 29 Antisense Inhibition of Rat Diacylglycerol Acyltransferase 1Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the ratdiacylglycerol acyltransferase 1 RNA, using published sequenceinformation (GenBank accession number AF296131.1, incorporated herein asSEQ ID NO: 154). The compounds are shown in Table 10. “Target site”indicates the first (5′-most) nucleotide number on the particular targetsequence to which the compound binds. All compounds in Table 10 arechimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of ten 2′-deoxynucleotides, whichis flanked on both sides (5′ and 3′ directions) by five-nucleotide“wings”. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides.The internucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

The compounds were analyzed for their effect on rat diacylglycerolacyltransferase 1 mRNA levels by quantitative real-time PCR as describedin other examples herein. Probes and primers to rat diacylglycerolacyltransferase 1 were designed to hybridize to a rat diacylglycerolacyltransferase 1 sequence, using published sequence information(GenBank accession number AF296131.1, incorporated herein as SEQ ID NO:154). For rat diacylglycerol acyltransferase 1 the PCR primers were:

forward primer: CAGACCAGCGTGGGCG (SEQ ID NO: 155)reverse primer: GAACAAAGAGTCTTGCAGACGATG (SEQ ID NO: 156) and the PCRprobe was: FAM-CGGCCACTGGGAGCTGAGGTG-TAMRA (SEQ ID NO: 157) where FAM isthe fluorescent reporter dye and TAMRA is the quencher dye. Rat targetgene quantities were normalized by quantifying total RNA usingRiboGreen™ RNA quantification reagent.

Data are averages from three experiments in which rat primaryhepatocytes were treated with 50 nM of the antisense oligonucleotides ofthe present invention. Data, shown in Table 10, are presented as percentinhibition normalized to untreated control samples.

TABLE 10Inhibition of rat diacylglycerol acyltransferase 1 mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET SEQ SEQ ID TARGET % ID ISIS # REGION NO SITE Sequence INHIB NO191726 5′UTR 11 1 ctcgtcgcggcccaatcttc 0 61 191733 Coding 11 261tcttgcagacgatggcacct 68 67 327788 Start Codon 154 24TCGCCCATGGCTTCGGCCCG 0 158 327789 Coding 154 44 AGCTTCCCGCGCCTCCGCGG 0159 327790 Coding 154 61 GGTCCTGCGACGCCGAGAGC 0 160 327791 Coding 154 82CTGGACGGAAACCCGCGAGC 0 161 327792 Coding 154 103 TACCTTGGGCCCACTACCTC 0162 327793 Coding 154 121 TCGCACCTCGTCCTCTTCTA 0 163 327794 Coding 154170 GAGCCGGCGCGTCACCCCCG 0 164 327795 Coding 154 191TATGGGCTGGAGCCGGAGCC 0 165 327796 Coding 154 196 CCGGGTATGGGCTGGAGCCG 0166 327797 Coding 154 225 TCGCCCACGCTGGTCTGCCG 63 167 327798 Coding 154248 GGCACCTCAGCTCCCAGTGG 64 168 327799 Coding 154 282CTGTCTGAGCTGAACAAAGA 0 169 327800 Coding 154 309 AGGATACCACGGTAATTGCT 0170 327801 Coding 154 318 CACCAATTCAGGATACCACG 0 171 327802 Coding 154345 GCATTACTCAGGATCAGCAT 0 172 327803 Coding 154 359CTAAAGATAACCTTGCATTA 0 173 327804 Coding 154 374 ACTTGATAAGATTCTCTAAA 0174 327805 Coding 154 389 CCACCAGGATGCCATACTTG 0 175 327806 Coding 154393 GGATCCACCAGGATGCCATA 0 176 327807 Coding 154 415AAACAGAGACACCACCTGGA 0 177 327808 Coding 154 463 GGATGCAATGATCAAGCATG 0178 327809 Coding 154 477 ACAATAAAGATATTGGATGC 0 179 327810 Coding 154499 CTTCTCAATCTGAAATGTAG 0 180 327811 Coding 154 504AGGCGCTTCTCAATCTGAAA 0 181 327812 Coding 154 527 GCTCTGTCAGGGCACCCACT 0182 327813 Coding 154 537 AGCCCCATCTGCTCTGTCAG 20 183 327814 Coding 154552 ACCACATGTAGCAGCAGCCC 17 184 327815 Coding 154 579GGGAAGCAGATAATTGTGGC 0 185 327816 Coding 154 594 AAGGCCACAGCTGCTGGGAA 0186 327817 Coding 154 607 AGACTCAACCAGTAAGGCCA 25 187 327818 Coding 154616 TGGAGTGATAGACTCAACCA 2 188 327819 Coding 154 649GGAGTATGATGCCAGAGCAA 0 189 327820 Coding 154 661 GAGGAAGATGATGGAGTATG 0190 327821 Coding 154 679 CCGGTAGGAAGAAAGCTTGA 0 191 327822 Coding 154709 CCTTCGCTGGCGGCACCACA 29 192 327823 Coding 154 726ACAGCTTTGGCCTTGACCCT 0 193 327824 Coding 154 744 ACCTTCTTCCCTGCAGACAC 0194 327825 Coding 154 758 CAGCAGCCCCACTGACCTTC 6 195 327826 Coding 154779 GATAGCTTACAGTGTTCTGG 0 196 327827 Coding 154 797GGTAGGTCAGGTTGTCCGGA 0 197 327828 Coding 154 806 AGAGATCTCGGTAGGTCAGG 0198 327829 Coding 154 819 AAGATGAAGTAATAGAGATC 0 199 327830 Coding 154833 ACAAAGTAGGAGCAAAGATG 0 200 327831 Coding 154 849AAGTTGAGTTCATAACACAA 0 201 327832 Coding 154 912 AAAAAGAGCATCTCAAGAAC 0202 327833 Coding 154 934 CAGCCCCACTTGAAGCTGGG 0 203 327834 Coding 154949 CATCCACTGCTGGATCAGCC 0 204 327835 Coding 154 970GGAGTTCTGGATAGTAGGGA 0 205 327836 Coding 154 981 AAGGGCTTCATGGAGTTCTG 0206 327837 Coding 154 991 CATGTCCTTGAAGGGCTTCA 0 207 327838 Coding 1541030 CGCCAGCTTTAAGAGACGCT 0 208 327839 Coding 154 1103GCTCTGCCACAGCATTGAGA 0 209 327840 Coding 154 1131 TAGAACTCGCGGTCTCCAAA 0210 327841 Coding 154 1162 GGTGACAGACTCAGCATTCC 0 211 327842 Coding 1541186 GATATTCCAGTTCTGCCAAA 0 212 327843 Coding 154 1212TGTCTGATGCACCACTTGTG 0 213 327844 Coding 154 1271 AGACCCCAGTCCTGGCCATC22 214 327845 Coding 154 1299 TACTCATGAAAGAAAGCTGA 0 215 327846 Coding154 1351 CATTGCTGTGAATGCCCAAA 0 216 327847 Coding 154 1380ACAATCCAGGCCAGTGGGAC 0 217 327848 Coding 154 1414 TGCATTGCCATAGTTCCCTT 0218 327849 Coding 154 1442 GCCCAATGATGAGTGTCACC 0 219 327850 Coding 1541477 GTAGTCGTGGACATACATGA 0 220 327851 Stop Codon 154 1524TTGGCAGTAGCTCATGCCCC 0 221 327852 Stop Codon 154 1531CTGGCCTTTGGCAGTAGCTC 12 222 327853 3′ UTR 154 1562 CCTCCAGAACTCCAGGCCCA55 223 327854 3′ UTR 154 1637 ATCCCCAAGAGCAGGAGTAG 0 224 327855 3′ UTR154 1670 CCCAGCACTGGCTCAACCAG 0 225 327856 3′ UTR 154 1702TTGATATCCTAAGCCCCTGG 0 226 327857 3′ UTR 154 1727 TTTTTTTTTTTTAGATAGCT 0227

As shown in Table 10, SEQ ID NOs 67, 167, 168, 183, 184, 187, 192, 214,222 and 223 demonstrated at least 10% inhibition of rat diacylglycerolacyltransferase 1 in this assay.

SEQ ID NOs 61 and 67 are cross-species antisense oligonucleotides thattarget both mouse and rat diacylglycerol acyltransferase 1.

Example 30 Antisense Inhibition of Rat Diacylglycerol Acyltransferase 1by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap: Dose Response

In a further embodiment, six oligonucleotides were selected for furtherinvestigation in a dose response experiment in rat primary hepatocytes.Rat primary hepatocytes were treated with 1, 5, 10, 25, 50 and 100 nM ofISIS 191733 (SEQ ID NO: 67), ISIS 327798 (SEQ ID NO: 168), ISIS 327814(SEQ ID NO: 184), ISIS 327817 (SEQ ID NO: 187), ISIS 327822 (SEQ ID NO:192), ISIS 327844 (SEQ ID NO: 214) and ISIS 327853 (SEQ ID NO: 223).Untreated cells served as a control. Target mRNA levels were measured byreal-time PCR as described in other examples herein. Data, presented inTable 11, are the average of three experiments and are normalized tountreated control samples.

TABLE 11 Inhibition of rat diacylglycerol acyltransferase 1 by chimericphosphorothioate oligonucleotides: dose response Dose of oligonucleotideSEQ ID 5 10 25 50 100 200 ISIS # NO % Inhibition 191733 67 20 53 77 9197 99 327798 168 0 13 68 88 96 98 327814 184 0 5 37 72 80 89 327817 1870 0 0 57 76 87 327822 192 0 32 52 73 88 95 327844 214 0 0 17 66 71 87327853 223 0 0 48 70 80 92

As demonstrated in Table 11, all 7 antisense oligonucleotides testedwere able to inhibit the expression of diacylglycerol acyltransferase 1in a dose-dependent manner.

All documents referenced in this specification are incorporated byreference.

1. A compound comprising a modified oligonucleotide 8 to 80 nucleobasesin length targeted to a nucleic acid molecule encoding diacylglycerolacyltransferase 1, wherein said compound specifically hybridizes withsaid nucleic acid molecule (SEQ ID NO: 4) encoding diacylglycerolacyltransferase 1 and inhibits the expression of diacylglycerolacyltransferase
 1. 2. The compound of claim 1, wherein the modifiedoligonucleotide is 12 to 50 nucleobases in length.
 3. The compound ofclaim 2, wherein the modified oligonucleotide is 15 to 30 nucleobases inlength.
 4. (canceled)
 5. The compound of claim 1, wherein the modifiedoligonucleotide is a single-stranded oligonucleotide.
 6. The compound ofclaim 1, wherein the modified oligonucleotide is a DNA oligonucleotide.7. The compound of claim 1, wherein the modified oligonucleotide is anRNA oligonucleotide.
 8. The compound of claim 1, wherein the modifiedoligonucleotide is a chimeric oligonucleotide.
 9. The compound accordingto claim 8, wherein said chimeric oligonucleotide is 20 nucleotides inlength, comprising ten 2′-deoxynucleotides, flanked on each side by five2′-methoxyethyl nucleotides, wherein the internucleoside linkages arephosphorothioate, and all cytidine residues are 5-methylcytidines. 10.The compound of claim 1, wherein at least a portion of said compoundhybridizes with RNA to form an oligonucleotide-RNA duplex.
 11. Thecompound of claim 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 70% complementary within a nucleic acidmolecule encoding diacylglycerol acyltransferase 1 (SEQ ID NO: 4). 12.The compound of claim 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 80% complementary within a nucleic acidmolecule encoding diacylglycerol acyltransferase 1 (SEQ ID NO: 4). 13.The compound of claim 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 90% complementary within a nucleic acidmolecule encoding diacylglycerol acyltransferase 1 (SEQ ID NO: 4). 14.The compound of claim 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 95% complementary within a nucleic acidmolecule encoding diacylglycerol acyltransferase 1 (SEQ ID NO: 4). 15.The compound of claim 1, wherein the modified oligonucleotide comprisesat least one modified sugar.
 16. The compound of claim 15, wherein themodified sugar comprises a 2′-O-methoxyethyl.
 17. The compound of claim1, wherein the modified oligonucleotide comprises at least one modifiedinternucleoside linkage.
 18. The compound of claim 1, wherein themodified oligonucleotide comprises at least one modified nucleobase.19-63. (canceled)
 64. The compound of claim 1, wherein the nucleobasesequence of the modified oligonucleotide is at least 99% complementarywithin a nucleic acid molecule encoding diacylglycerol acyltransferase 1(SEQ ID NO: 4).
 65. The compound of claim 1, wherein the nucleobasesequence of the modified oligonucleotide is 100% complementary within anucleic acid molecule encoding diacylglycerol acyltransferase 1 (SEQ IDNO: 4).
 66. The compound of claim 15, wherein the modified sugar is abicyclic sugar.
 67. The compound of claim 17, wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 68. The compoundof claim 18, wherein the modified nucleobase is a 5-methylcytosine. 69.A composition comprising the compound of claim 1 and a pharmaceuticallyacceptable carrier or diluent.